KARSTENIA 39 (1999) HINTIKKA & NIEMI: RUSSULA A ND CORTINARIUS 39

Karstenia 39:39-41, 1999

Aseptic culture of slowly growing mycorrhizal Russula and Cortinarius species

VEIKKO HINTIKKA and KAROLIINA NIEMI

HINTIKKA, V. & NIEMI, K. 1999: Aseptic culture of slowly-growing mycorrhizal Russula and Cortinarius species. - Karstenia 39: 39-41. Helsinki. ISSN 0435-3402 Aseptic cultures of several Cortinarius and Russula species were obtained by transfer­ ring first a piece of the context of the sporocarp aseptically on malt extract agar, and after 2-4 weeks, if not contaminated with bacteria or molds, into liquid MMN medium. It took several months before mycelia slowly started to grow in the solution. When cultivated on MMN- agar media, most species grew only fe\ millimetre/month, re­ markably less than many other ectomycorrhizal species tested. Fungal species reacted very specifically to different sources. The slow growth rate of Russula and Cortinarius species may be related to specific requirements (nutrients and/or itamins) not yet known. Possible role of hidden infection of bacteria is also discussed. Key words: aseptic culture, Cortinarius, growth rate, Russula

Ve ikko Hintikka, Department of Plant Biolog) ~ PO. Box 27, F/N-00014 University of Helsinki, Finland. Karoliina Niemi ':', Department of Plant Biology, PO. Box 27, F/N- 00014 University of Helsinki, Finland. ''' Permanent address: Departm nt of Ecology and Environmen­ tal Science, University of Kuopio,PO. Box 1627, 70211 Kuopio.

Introduction Methods and results Although Russula and Cortinarius species, ec­ A small piece of the context of the basidiocarp tomycorrhizal symbionts of many tree species, was taken aseptically and transferred to 1% malt often play a dominant role in the macromycete extract agar slant and kept there 2-4 weeks to flora of Nordic forests, relatively little is known of ensure that the piece was free from bacterial or their exact role in the ecosystem. mold infections. During this period, no clear Attempts to obtain aseptic cultures have been growth was seen. From agar medium the piece largely unsuccessful, and only a few synthesis was immersed into modified MelinNorkrans experiments have been carried out (Agerer 1986, (MMN-) solution in 100 ml Erlenmeyer flasks. In Taylor & Alexander 1989, Ali & Jackson 1989), positive cases, a sparse mycelium started to compared with e.g. bolets and Amanita species grow from the piece in 2-3 months. In all cases which grow easily on cultural media. In this paper the liquid remained clear and evidently free from we describe the method by which aseptic cul­ bacterial infections. After one year, there was a tures of several Russula and Cortinarius species relatively large mycelium piece and after one and were obtained. a half year the mycelium filled up the whole liquid 40 HINTIKKA & NIEMI: RUSSULA AND CORTINARIUS KARSTENIA 39 (1999) medium. The mycelia grew largely submerged, L-glutamine was added into 60-70° agar media. and had no clamp connections. The final concentration of inositol and L­ The radial growth of the mycelia was studied glutamine was 0.5% and that of other compo­ by cultivating them on MMN- agar in Petri dish­ nents 1%. Radial growth was measured after 28 es. The growth was compared to that of some days. other ectomycorrhizal species after 34 days of The species reacted specifically to different cultivation. During the experiment the fastest carbon sources (Table 2). Both Russula species fungi grew up to 40 mm, in contrast to slower preferred fructose 1,6-P, gentiobiose and also ones, which grew only few millimetres (Table 1). glutamine to glucose and sucrose. To Corti­ The results indicate that there is a continuous narius argutus fungal sugars trehalose and man­ series from rapidly growing mycorrhizal species, nitol were slightly better carbon sources than such as Paxillus involutus and some Suillus glucose and clearly better than sucrose. In C. species, to very slowly growing Russula and anomalus, no clear difference between these Cortinarius species. sugars was observed. L-glutamine was not a suitable carbon source for Cortinarius species. Surprisingly, both Russula delica and Corti­ narius argutus grew well without any added car­ Table 1. Radial growth (mm) of certain ectomy­ bon source, although the mycelium was very corrhizal fungi on MMN- agar after 34 days. thin. Russula xerampelina grew only on treha­ lose and Russula paludosa on maltose media, Paxillus involutus (Batsch : Fr.) Fr. 45 mm both less than 1 mm during 28 days (data not Suillus bovinus (L. :Fr.) Roussel 40 mm shown). Cortinarius mucosus produced thin S. luteus (L. : Fr.) Roussel 37 mm Suillus granulatus (L. : Fr.) Roussel 37 mm aerial rhizomorphs on cellobiose medium. Amanita rubescens (Pers. : Fr.) S.F.Gray 26 mm Cortinarius argutus Fr. 17 mm Boletus badius (Fr.) Fr. 13 mm Cortinarius mucosus (Bull.: Fr.) Kickx 12 mm C. anomalus (Fr. : Fr.) Fr. 8mm Table 2. The radial growth of four ectomycorrhiz- Leccinum versipelle (Fr.) Snell 8 mm Tricholoma album (Fr.) Kumm. 8mm al fungi on MMN- agar supplemented with dif- Lactarius mitissimus (Fr.) Fr. 7 mm ferent carbon sources (1 %). The radial growth Russula vinosa Lindbl. 7 mm (mm) was measured after 28 days. Amanita citrina (Schaeff.)Pers. 6mm Russula delica Fr. 6mm Russula Russula Cortinarius Cortinariu R. paludosa Britz. 6mm vinosa delica argutus anomalus Lactarius torminosus (Schaeff. : Fr.) Pers. 5 mm Russula xerampelina (Schaeff.) Fr. 3 mm no addition 0 10 10 I 2mm Cortinarius cumatilis Fr. glucose 2 2 9 4 I mm Russula foetens Pers. : Fr. glucose 1-P 0 6 1 0 R. grata Britz (R. laurocerasi) 1 mm sucrose 2 + 5 3 (L. Cortinarius violaceus :Fr.) S.F.Gray <1 mm maltose 2 4 2 2 fructose 1,6-P 5 4 3 0.5 trehalose 3 2 11 2 sellobiose 3 2 9 3 The influence of different carbon sources, gentiobiose 4 6 5 3 malt extract 0.2 1 9 4 sugars and an amino acid L-glutamine, on the ra­ mannitol 0 4 12 5 dial growth of the mycelia was studied by culti­ inositol 0,5'7c I 0 0 0 vating the mycelia on agar media containing 500 glutamine 0.5% 5 9 0 0 (+) mgKHl0 ,500mgNH Cl, 100mgMgS0 , 10mg sorbitol 0 I 0 4 4 4 xylitol 2 2 6 + , , MnS04 10 mg ZnS04 1 mg thiamine, 1 rnl1% Fe­ citrate solution and 10.0 g agar in 1000 ml deion­ ised water. After the medium had been auto­ claved, it was poured into sterile erlenmeyer flasks (100 rnl), and filter-sterilized (Millipore size 0.22 11m) aqueous solution of a single sugar or KARSTENIA 39 (1999) HINTIKKA & NIEMI: RUSSULA AND CORTINARIUS 41

Discussion References The slow growth rate of many ectomycorrhizal Agerer, R. 1986: Studies on ectomycorrhizae III. My­ fungi is well known (Smith & Read 1997), espe­ corrhizae formed by four fungi in the genera Lactarius and Russula on spruce.- Mycotaxon 27: 159. cially when compared to saprophytic wood-de­ Ali, N. & Jackson, R.M. 1989: Stimulation of germina­ composing fungi. If compared with the expan­ tion of spores of some ectomycorrhizal fungi by oth­ sion rate of basidiocarps, the growth rate of the er micro-organisms. - Mycol. Res. 93: 1821 -86. mycelium is surprisingly low. For example, in this Danell, E. & Fries, N. 1993: Methods for isolation of Cantharellus cibarius species, and the synthesis of ec­ study the mycelium of Russula paludosa grew tomycorrhizae with Picea abies. - Mycotaxon 38 : only 6 mm in 34 days, while caps of the same 141-148. species have been reported to extend up to 34 Erland, S. & Finlay, R.D. 1992: Effects of temperature mm in a single day in suitable conditions (Hiukka and incubation time on the ability of three ectomyc­ 1978). orrhizal fungi to colonize P. sylvestris roots. - Mycol. Res. 96: 270-272. Russula and Cortinarius are mycorrhizal fun­ Hiukka, E. 1978: Helttasienten itioemien kasvunopeu­ gi, which appear mostly in the late stage of forest desta (On the growth rate of fruitbodies of agarics). - succession. Growth rate in vitro has been sug­ Sienilehti 30: 40-42. gested to relate to successive changes in ecto­ Sarvas, M. I 976: Pseudomonas ja muut viihiin patogee­ mycorrhizal colonization of pine roots (Erland & niset gram-negatiiviset sauvat. - In: Miikelii, 0., Miikelii, P.H., Wager, 0. & Vaheri, A. (eds), Liiiike­ Finlay 1992). The slow growth rate of both Rus­ tieteellinen mikrobiologia: 255-272. Duodecim Hel­ sula and Cortinarius species on aseptic cultures sinki. might be due to specific requirements, such as Smith, S.E. & Read, D.J. 1997: Mycorrhizal symbiosis.­ nutrients and/or vitamins, not yet known. Be­ Academic Press, London. 605 pp. Taylor, A.F.S. & Alexander, I.J. 1989: Ectomycorrhizal cause both Russula delica and Cortinarius ar­ synthesis with an isolate of Russula aeruginea.- My­ gutus grew as well or even better on the control col. Res. 92: 103- 107. media (no carbon source added) than on the me­ Varese, G.C., Portinaro, S., Trotta, A., Scanneri, S., Lup­ dia supplemented with several different carbon pi-Mosca, A.M. & Martinotti, M.G. 1996: Bacteria associated \ ith Suillus grevillei sporocarps and ecto­ sources, the change of energy source is not the mycorrhizae and their effects on in vitro growth of probable solution for better growth. Instead, it the mycobiont. - Symbiosis 21: 129- 147. would be important to study possible roles of micronutrients and different forms of nitrogen in Received on 8 June 1999 mycelial growth. Bacteria associated with basidiocarps have been reported to inhibit the growth of mycelia of, for example, Cantharellus cibarius (Danell & Fries 1993) and Suillus grevillei (Varese et al. 1996). Also in this study, hidden infection of bac­ teria might reduce the growth of mycelia, al­ though cultivation on 0.5% streptomycin agar did not result in any better growth of the mycelia. However, it should be noted that Pseudomonas bacteria, which occur in plant tissues and in soil, are very resistant to antibiotics (Sarvas 1976).